At a fundamental level, advances in biomaterials research are driven by the ability to elicit desired cell behaviors through the development of instructive biomaterial surfaces. Rational design of novel biomaterials that can successfully manipulate cell behavior toward a specific function requires both identification of instructive cues and a mechanistic understanding of how these cues alter cellular function. However, determining the specific combination of cues to induce a precise set of cellular behaviors leading to a predictable functional outcome remains challenging. The challenge stems from an incomplete understanding of the mechanisms used by cells to detect and respond to environmental cues, particularly physical cues such as confinement of cell shape or stiffness of the extracellular environment. Physical cues are detected through mechanotransduction, a poorly understood process through which cells convert physical stimuli into biochemically detectable signals. Recent advances in the study of mechanotransduction have isolated a key role for the focal adhesion protein vinculin, an important linkage in the mechanical connections between the extracellular environment and the force-generating actin cytoskeleton. This proposal seeks to evaluate the hypothesis that cells sense diverse changes in the physical nature of the cell-biomaterial interface through distinct mechanical loading of vinculin, leading to the activation of cell signaling pathways. Specifically, we will study the effects of cell shape, cell size, and extracellular proteins on force-sensitive signal activation. The proposed work is relevant to the mission of NIH as it will increase the fundamental understanding of how cells sense and respond to the physical aspects of their surroundings. This will lay the foundation for advances in biomaterials design as well as aid efforts to understand disease states associated with defects in the physical nature of the cellular environment, such as cancer and excessive tissue fibrosis.